RESUMEN
Two distinct species of rat angiotensinogen (A-1 and A-2) were purified from plasma of nephrectomized rats by combining ammonium sulfate fractionation, chromatography on Blue Sepharose and SP-Sephadex, gel filtration and preparative polyacrylamide gel electrophoresis. Separation of the two species was accomplished in the SP-Sephadex chromatography step, A-1 eluting before A-2. The two angiotensinogen species had identical electrophoretic mobilities on analytical polyacrylamide gel electrophoresis, but differed in their apparent molecular weights as obtained by SDS-gel electrophoresis (A-1, Mr 60 000; A-2, Mr 56 400). In analytical isoelectric focusing each species displayed a characteristic double band with isoelectric points of 4.54 and 4.60 for A-1, and 4.69 and 4.76 for A-2. These physicochemical differences can be accounted for by the difference in carbohydrate content: A-1, when compared to A-2, had a higher content of sialic acid (5.0 and 2.1 mol/mol), neutral hexoses (10.2 and 5.9 mol/mol) and aminohexoses (10.5 and 7.0 mol/mol, respectively). Antiserum raised against rat angiotensinogen crossreacted completely with both angiotensinogens. Both species could also be isolated from plasma of non-nephrectomized rats, which indicates that they may be present under physiological conditions. The physiological significance of the occurrence of these species of angiotensinogen is still unknown.
Asunto(s)
Angiotensinógeno/aislamiento & purificación , Angiotensinas/aislamiento & purificación , Angiotensinógeno/sangre , Animales , Inmunodifusión , Cinética , Masculino , Peso Molecular , Nefrectomía , Ratas , Ratas EndogámicasRESUMEN
1. Angiotensinogen (renin substrate) was purified from plasma of nephrectomized rats by a four step procedure using ammonium sulfate fractionation, chromatography on Blue Sepharose CL-6B and SP-Sephadex C-50, and gel filtration on Sephadex G-150. 2. The final preparation had a specific concentration of 9.3 microgram angiotensin I/mg (mean of six separate runs). The best preparation so far obtained contains 14.6 microgram angiotensin I/mg protein, which represents a purity of 62%. 3. By sodium dodecyl sulfate disc electrophoresis an apparent molecular weight of 56,400, and by isoelectric focusing an isoelectric point of 4.85 has been determined. These properties of rat angiotensinogen are similar to those reported for human angiotensinogen.
Asunto(s)
Angiotensinógeno/sangre , Angiotensinas/sangre , Angiotensinógeno/aislamiento & purificación , Animales , Fenómenos Químicos , Química , Cromatografía en Gel , Electroforesis , Focalización Isoeléctrica , Masculino , Ratas , Ácidos Siálicos/análisisRESUMEN
1. Isorenin was purified 2000-fold from rat brain by a simple 3-step procedure involving affinity chromatography on pepstatinyl-Sepharose, The preparation appears as a homogenous protein in analytical polyacrylamide gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis indicated an apparent molecular weight of 45 000. Isoelectric focusing separated isoenzymes with isoelectric points at pH 5.45, 5.87, 6.16 and 7.05. 2. The enzyme generates antiotensin I from tetradecapeptide (pH optimum 4.7) and from sheep angiotensinogen (pH optima 3.9 and 5.5). The rate of angiotensin I formation from tetradecapeptide was 30 000 times higher than that from sheep angiotensinogen. The enzyme has acid protease activity at pH 3.2 with hemoglobin as the substrate and pepstatin is a potent inhibitor of the enzyme with a Ki of less than 10(-9) M. 3. The properties of the enzyme strongly suggest that it is identical with cathepsin D.
Asunto(s)
Encéfalo/enzimología , Endopeptidasas/metabolismo , Angiotensina I/biosíntesis , Angiotensinógeno/metabolismo , Animales , Endopeptidasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Masculino , Peso Molecular , Pepstatinas/farmacología , RatasRESUMEN
1. Renin was purified 30 000-fold from rat kidneys by chromatography on DEAE-cellulose and SP-Sephadex, and by affinity chromatography on pepstatinyl-Sepharose. 2. The enzymatic properties of isorenin from rat brain, pseudorenin from hog spleen, cathepsin D from bovine spleen, and renin from rat kidneys were compared: Isorenin, pseudorenin and cathepsin D generate angiotensin from tetradecapeptide renin substrate with pH optima around 4.9, renin at 6.0. With sheep angiotensinogen as substrate, isorenin, pseudorenin and cathepsin D have similar pH profiles (pH optima at 3.9 and 5.5), in contrast to renin (pH optimum at 6.8). 3. The angiotensin-formation from tetradecapeptide by isorenin, pseudorenin and cathepsin D was inhibited by albumin, alpha-and beta-globulins. These 3 enzymes have acid protease activity at pH 3.2 with hemoglobin as the substrate. Renin is not inhibited by proteins and has no acid protease activity. 4. Renin generates angiotensin I from various angiotensinogens at least 100 000 times faster than isorenin, pseudorenin or cathepsin D, and 3000 000 times faster than isorenin when compared at pH 7.2 with rat angiotensinogen as substrate. 5. The 3 'non-renin' enzymes exhibit a high sensitivity to inhibition by pepstatin (Ki less than 5.10(-10) M), in contrast to renin (Ki approximately 6-10(-7) M), at pH 5.5. 6. It is concluded from the data that isorenin from rat brain and pseudorenin from hog spleen are closely related to, or identical with cathepsin D.
Asunto(s)
Angiotensinas/biosíntesis , Catepsinas/metabolismo , Renina/metabolismo , Albúminas/farmacología , Angiotensinógeno/metabolismo , Animales , Bovinos , Endopeptidasas , Cinética , Masculino , Pepstatinas/farmacología , Ratas , PorcinosRESUMEN
Rat plasma contains two distinct forms of angiotensinogen (Ao-1 and Ao-2) that can be found in single animals in a distinct ratio. The ratio of Ao-1 to Ao-2 was determined by separation of Ao-1 and Ao-2 from 1 ml of plasma from individual rats on an SP-Sephadex C-50 column. Plasma from rats of three different strains, Wistar, Wistar-Kyoto (WKY), and spontaneously hypertensive rats (SHR), was investigated. In Wistar rats native plasma contained Ao-1 and Ao-2 in a ratio of 2.6:1. Twenty-four hours after nephrectomy, which increased the total Ao content 4.1-fold, this ratio was changed to 1.1:1. In native WKY and SHR the ratio of the two forms was similar to that in Wistar rats: 2.4:1 and 2.8:1, respectively. After nephrectomy the ratio of Ao-1 to Ao-2 was changed to 1.1:1 and 0.78:1 in WKY and SHR, respectively, while the total Ao content increased 4.9-fold and 8.2-fold in the two strains. Endogenous plasma renin inactivated the two forms of Ao, with a Km of 4.0 +/- 0.46 and 3.7 +/- 0.43 microM and a Vmax of 176 +/- 15.5 and 155 +/- 12.7 nM/hr, respectively. These results suggest that 1) Ao-1 and Ao-2 are synthesized in equimolar amounts, 2) the clearance of Ao-2 is faster than that of Ao-1 in control rats, and 3) under conditions of stimulated synthesis (i.e., after nephrectomy), the plasma content of Ao-2 increases faster than that of the more highly glycosylated form, Ao-1.
Asunto(s)
Angiotensinógeno/sangre , Riñón/fisiología , Animales , Glicosilación , Nefrectomía , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas , Ratas Endogámicas WKY , Sistema Renina-AngiotensinaRESUMEN
The cleavage of synthetic tetradecapeptide renin substrate has been used to infer the presence of renin in the walls of isolated blood vessels; however, the conversion of natural angiotensinogen to angiotensin in isolated blood vessels has not been reported. We studied the release of angiotensinogen and the formation of angiotensins in a bloodless, perfused, isolated hind limb preparation of the rat. Perfusion with a modified Tyrode's solution resulted in spontaneous release of 4.7 +/- 1.5 pmol per 30 minutes of angiotensinogen as measured directly by radioimmunoassay. Western blot further identified the released material as angiotensinogen. Spontaneous release of angiotensins I and II was demonstrated by high performance liquid chromatography and radioimmunoassay. When highly purified rat angiotensinogen was added to the perfusate, release of angiotensin II was increased 14-fold compared with saline infusion. Captopril (10 mumol/L) inhibited angiotensinogen-induced angiotensin II release by 67% and led to an increase in angiotensin I release by 301%. Bilateral nephrectomy 24 hours before the experiments reduced basal angiotensin release below the detection limit and blunted angiotensinogen-induced angiotensin II formation by 95%. We conclude that active renin is present in the vessel wall and interacts with its natural substrate to form angiotensin peptides. Our data support the notion that the bulk of vascular renin is taken up from the circulation.
Asunto(s)
Angiotensina II/metabolismo , Angiotensinógeno/metabolismo , Vasos Sanguíneos/metabolismo , Angiotensina I/metabolismo , Angiotensinógeno/farmacología , Animales , Captopril/farmacología , Miembro Posterior/irrigación sanguínea , Técnicas In Vitro , Masculino , Perfusión , Ratas , Ratas Sprague-DawleyRESUMEN
To examine and characterize the vascular renin--angiotensin system in low-renin models of renal hypertension with and without the presence of overt renal insufficiency, we studied the formation and metabolism of angiotensin in isolated perfused rat hindquarter preparations. Rats with 5/6 nephrectomy (5/6NX) and rats with one-kidney, one clip (1K1C) hypertension were compared to sham operated (sham) animals. Angiotensin peptides in plasma or perfusate were characterized by high-performance liquid chromatography and radioimmunoassay (RIA). Plasma angiotensin II was lower, and blood pressure was higher in both experimental groups, compared to sham animals. Plasma angiotensinogen, measured by both direct and indirect RIA, was increased in both experimental groups. The spontaneous release of angiotensin I and angiotensin II from perfused hindquarters did not differ between the groups. Angiotensin I conversion was not different in 5/6NX or 1K1C groups compared with controls. Furthermore, angiotensin conversion was completely inhibited by captopril (1 mumol/l) in all groups. Renin-induced angiotensin release was significantly increased in 5/6NX as compared with sham rats, whereas there was no difference in renin-induced angiotensin release between 1K1C and sham animals. Angiotensin II degradation was significantly attenuated in 5/6NX rats when compared with sham rats (27.6% versus 53.9%, respectively, P less than 0.05) but was unaltered in 1K1C rats. Thus, in chronic uremic hypertension, renin-induced angiotensin formation was increased in the face of decreased angiotensin II degradation. These data suggest that vascular angiotensin may contribute to the elevated blood pressure observed in chronic renal failure. In 1K1C rats, vascular angiotensin formation and metabolism was unchanged despite suppressed plasma angiotensin II.
Asunto(s)
Angiotensina II/biosíntesis , Vasos Sanguíneos/metabolismo , Hipertensión Renal/metabolismo , Hipertensión Renovascular/metabolismo , Sistema Renina-Angiotensina/fisiología , Renina/fisiología , Uremia/metabolismo , Angiotensina II/metabolismo , Angiotensinógeno/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Miembro Posterior , Masculino , Radioinmunoensayo , Ratas , Ratas EndogámicasRESUMEN
OBJECTIVE: We have reported that bradykinin (BK) excretion is increased in severely diabetic rats, independent of the activity of the main renal kinin-forming enzyme, true kallikrein (KLK). To further investigate the relationship between renal BK excretion and renal KLK in diabetes we studied the regulation of the renal kallikrein-like gene, rat kallikrein 7 (rKLK7), as well as of the KLK encoding gene, rKLK1, in streptozotocin-induced (STZ) diabetic rats. METHODS: Experiments were performed in STZ-induced diabetic male Wistar rats and their non-diabetic controls (n = 7 each group). Twelve weeks after STZ injection, urinary KLK activity, glomerular filtration rate and total protein excretion were determined. After extraction of total renal cortical RNA, specific oligonucleotides were used to generate a reverse transcription-polymerase chain reaction (RT-PCR) products of renal cortical rKLK1 and rKLK7 messenger (m)RNA. Southern blot analysis of these RT-PCR products were hybridized with appropriate gene-specific oligonucleotide probes. RESULTS: After 12 weeks, the rats showed hyperglycemia, proteinuria and a reduced glomerular filtration rate. Renal kininogenase was reduced, as indicated by a reduction in the expression of rKLK1, as well as of the KLK-related gene, rKLK7. CONCLUSIONS: Our data show that the expression of the two principal renal KLK genes is downregulated in the renal cortex of STZ-diabetic rats. We suggest that under severe diabetic conditions the rise in urinary BK excretion is not related to activation of the renal kinin-forming enzyme system.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Regulación de la Expresión Génica , Calicreínas/metabolismo , Corteza Renal/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Southern Blotting , Modelos Animales de Enfermedad , Tasa de Filtración Glomerular , Calicreínas/genética , Glomérulos Renales/metabolismo , Masculino , Sondas de Oligonucleótidos/química , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
The in vivo half-life of two differently glycosylated subforms of rat angiotensinogen (Ao), Ao-1 and Ao-2, was studied in native and nephrectomized rats, as well as in rats treated with lipopolysaccharides (LPS). The metabolic clearance rate of both 125I-labeled derivatives fits a two-compartment model using a non-linear curve fitting program (SCTFIT). Both forms show an initial rapid phase with a half-life of about 1 h. This clearance rate was not altered by either nephrectomy or LPS administration. In the intact rat the two forms were eliminated with a second slower phase half-life of 8.07 h (Ao-1) and 4.53 h (Ao-2), respectively. Following nephrectomy, in the absence of renin, the slower phase of clearance of both forms decreased (t1/2 Ao-1:9.77 h; t1/2 Ao-2: 10.50 h). Induction of low renin concentrations by LPS administration decreased the half-life of the more highly glycosylated form Ao-1 (t1/2: 6.93 h), while that of Ao-2 increased (t1/2: 5.07 h). Inactive angiotensinogen, des-AngI-Ao, of both forms demonstrated a faster metabolic clearance rate in native rats than that of the intact glycoproteins (t1/2 Ao-1: 5.68 h; t1/2 Ao-2: 3.97 h). Based on the plasma levels of these two angiotensinogen subtypes in normal rats, these data suggest a higher secretion rate by the liver for the more highly glycosylated form Ao-1 and a slower clearance rate by the kidney. In contrast, the less glycosylated form shows more rapid elimination by the kidney. Following nephrectomy both glycoproteins are secreted and eliminated in an equimolar ratio.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Angiotensinógeno/metabolismo , Lipopolisacáridos/farmacología , Animales , Cromatografía Líquida de Alta Presión , Glicosilación , Semivida , Radioisótopos de Yodo , Masculino , Nefrectomía , Radioinmunoensayo , Ratas , Ratas EndogámicasRESUMEN
Angiotensinogen (Ao), the precursor of the peptide hormone angiotensin, exists in two different forms in plasma, Ao-1 and Ao-2. The completely separated and purified molecules show distinct differences in sodium dodecyl sulfate (SDS)-disc electrophoresis and in analytical isoelectric focusing. Ao-1 is about 3500 Da heavier and contains more acid isoelectric points than Ao-2. In analytical isoelectric focusing Ao-1 displays five bands which overlap with two of the three bands of Ao-2. This difference in charge was eliminated by treatment with neuraminidase for 92 h at 37 degrees C. Thereafter both molecules display an identical-pattern two bands in isoelectric focusing. Treatment of these N-acetylneuraminic acid (NeuNAc)-free forms with renin leads to a shift of these double bands to a more acid pH, indicating heterogeneity within the protein structure. By affinity chromatography on concanavalin A (ConA)-Sepharose, both forms can be separated in three fractions. The analysis of these fractions and of their NeuNAc-free derivatives by SDS-disc electrophoresis, as well as by analytical isoelectric focusing, leads to the conclusion that Ao-1 contains two Asn-linked N-glycan residues with 4-8 mol of NeuNAc, while the smaller form, Ao-2, contains one carbohydrate residue with 2-4 mol of NeuNAc.
Asunto(s)
Angiotensinógeno/análisis , Carbohidratos/análisis , Animales , Electroforesis Discontinua , Focalización Isoeléctrica , Masculino , Neuraminidasa/metabolismo , Ratas , Ratas EndogámicasRESUMEN
The study investigates the change in angiotensinogen (Aogen), angiotensin I (AngI) and renin plasma concentration after nephrectomy and adrenalectomy. The aim of the study was to elucidate the mechanisms that are involved in the up regulation of the Aogen plasma levels after nephrectomy and the contribution of the adrenals. Rats were treated with the beta 1-selective adrenoceptor blocker, atenolol, and with the angiotensin antagonist, DuP 753 in order to inhibit renal renin release and to check whether the increase in plasma Aogen after nephrectomy is mediated by angiotensin (AngII), respectively. The plasma Aogen levels increase approx. 5-fold 24 h after nephrectomy. This increase is significantly reduced in the presence of atenolol. After nephrectomy plus adrenalectomy there is a maximal increase of 60% in plasma Aogen levels 8 h after surgery and a subsequent decline. In the presence of atenolol this increase is even smaller. In contrast after adrenalectomy the plasma Aogen levels continuously declined. In the presence of atenolol the plasma Aogen levels were approx. 20% higher at time 0 but declined with the same slope as after adrenalectomy without atenolol treatment. Treatment with DuP 753 caused an almost complete inhibition of the increase in Aogen plasma levels after nephrectomy. Significantly higher Aogen levels were found only after 24 h. At time 0, immediately after nephrectomy the plasma AngI levels were increased compared to the respective control rats. Significantly higher AngI values (P < 0.05) could also be observed in nephrectomized rats and in nephrectomized plus adrenalectomized rats at time 0 in the presence and absence of atenolol and DuP 753, respectively. In contrast after adrenalectomy alone the AngI levels at time 0, were not different from those of the controls. Subsequently the AngI levels increased at a similar rate as after adrenalectomy in the presence of atenolol. These findings suggest that the increase in plasma Aogen after nephrectomy is essentially mediated by AngII via an adrenal mechanism. It seems likely that this process is triggered by renin released during surgery. The increased renin release after adrenalectomy that is responsible for the increased degradation of Aogen seems not to be mediated by a sympathetic stimulation of the renal beta 1-adrenoceptors.
Asunto(s)
Adrenalectomía , Nefrectomía , Sistema Renina-Angiotensina , Angiotensina I/sangre , Angiotensinógeno/sangre , Animales , Masculino , Ratas , Ratas Wistar , Renina/sangreRESUMEN
In the present study effects of estrogens (natural estradiol and synthetic ethinyl estradiol) on liver derived proteins (angiotensinogen, IGF-I) were investigated in vivo in ovariectomized rats and in vitro in a rat hepatoma cell line (Fe33). The aim of this study was to establish both an animal and an in vitro model for quantification of the hepatic activity of given estrogenic compounds, and to study underlying mechanisms as regards the question of direct or indirect mode of estrogen action. In ovariectomized rats subcutaneous (s.c.)-treatment for 11 days with either estradiol (E2) or ethinyl estradiol (EE) (dose range 0.1-3 micrograms/animal/day) induced a comparable dose-dependent increase in uterine weight indicating a similar estrogenic potency of the two estrogens. Equipotency was also found as regards the effects on IGF-I plasma levels which dose-dependently decreased by about 50% at the highest dose tested (3 micrograms/animal/day). The decrease in IGF-I serum levels was accompanied by a significant 40% decrease in liver IGF-I mRNA. In contrast angiotensinogen plasma levels were affected only by EE (60% increase for the 3 micrograms/animal/day dose) but not by E2. When rats, in addition to ovariectomy, were also hypophysectomized (substituted with human growth hormone and dexamethasone) angiotensinogen again increased by 80% upon administration of 3 micrograms/animal/day EE, whereas IGF-I remained unaffected by EE. In a rat hepatoma cell line (Fe33) which is stably transfected with an estrogen receptor expression plasmid, 10 nmol/l EE for 24 h caused a 2.4-fold increase in angiotensinogen mRNA level. We conclude from our studies that estrogen effects on angiotensinogen serum levels in the rat are direct effects via the hepatic estrogen receptor, whereas estrogen effects on IGF-I serum levels are indirect effects, the primary target of estrogen action being probably the pituitary. The changes in angiotensinogen serum levels in the rat model are comparable to the situation in humans indicating the rat model and the Fe33 model to be useful tools to study the hepatic activity of estrogenic compounds.
Asunto(s)
Angiotensinógeno/biosíntesis , Estradiol/farmacología , Etinilestradiol/farmacología , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Hígado/efectos de los fármacos , Angiotensinógeno/genética , Animales , Secuencia de Bases , Carcinoma Hepatocelular/metabolismo , Relación Dosis-Respuesta a Droga , Estradiol/administración & dosificación , Etinilestradiol/administración & dosificación , Femenino , Hipofisectomía , Factor I del Crecimiento Similar a la Insulina/genética , Cinética , Hígado/metabolismo , Neoplasias Hepáticas/metabolismo , Datos de Secuencia Molecular , Tamaño de los Órganos/efectos de los fármacos , Ovariectomía , ARN Mensajero , Ratas , Ratas Wistar , Células Tumorales Cultivadas , Útero/anatomía & histologíaRESUMEN
Angiotensinogen was localized in 3 cell types in brain using immunohistochemical methods. These locations included subpopulations of neurons in nuclei that co-stain for angiotensin II, subpopulations of astrocytes that make putative contacts with brain microvessels, and cells of the choroid plexus. These findings are consistent with multiple functions for brain angiotensinogen as a precursor for neuronal angiotensin II and as a potential source for angiotensin II that is locally produced in the brain.
Asunto(s)
Angiotensinógeno/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Angiotensina II/metabolismo , Animales , Plexo Coroideo/metabolismo , Hipotálamo/metabolismo , Técnicas para Inmunoenzimas , Masculino , Neuronas/metabolismo , Ratas , Ratas Endogámicas WKYRESUMEN
This study focuses on the renin-angiotensin system (RAS) in the cerebellar cortex and changes within this system after mechanically induced cerebellar injury. Using radioactive and non-radioactive in situ hybridization and immunocytochemistry angiotensinogen mRNA, angiotensinogen, angiotensin II and, for the first time, N-terminal angiotensin fragment (1-7) immunoreactivities, respectively, were demonstrated in the rat cerebellum. Angiotensinogen mRNA and angiotensinogen immunoreactivity (IR) were both present in glial cell populations of all layers, especially in the Purkinje and granular cell layers and within the cerebellar nuclei. Angiotensin II IR was demonstrated in glial cell populations in all layers using a monoclonal angiotensin II antibody, while with a polyclonal angiotensin II antiserum (Denise) some Purkinje cell bodies were labelled. After lesioning the cerebellar cortex mechanically by an injection cannula a strong increase in angiotensinogen gene expression as well as in angiotensin II and angiotensin (1-7) immunoreactivities were observed in the glial cell populations. Furthermore, putative Bergmann glial processes, as indicated from the morphological appearance became strongly angiotensin II and angiotensinogen immunoreactive in the region close to the mechanically induced lesion. It could inter alia be demonstrated for the first time using confocal laser microscopy of ANG II IR and GFAP IR that ANG II in vivo in the intact cerebellar cortex is present in astroglial processes in the molecular layer and presumably secreted into the extracellular space in form of small spheric bodies and/or taken up by other cell types. In contrast, the N-terminal fragment angiotensin (1-7) IR was restricted to the glial cell populations and appeared only after the lesion event. Thus, it is suggested that the cerebellar RAS shows marked changes in response to mechanically induced lesions. The expression of angiotensinogen as well as the production of angiotensinogen IR and angiotensin II like IR is even after mechanical lesion restricted to astrocytes, i.e., cerebellar astrocytes and putative Bergmann glial cells, and in case of immunoreactivities it spreads to the radially oriented Bergmann glial processes in the molecular layer.
Asunto(s)
Cerebelo/química , Cerebelo/lesiones , Plasticidad Neuronal , Sistema Renina-Angiotensina , Angiotensina I/análisis , Angiotensina II/análisis , Angiotensinógeno/análisis , Animales , Cerebelo/metabolismo , Proteína Ácida Fibrilar de la Glía/análisis , Inmunohistoquímica , Hibridación in Situ , Masculino , Microscopía Confocal , Neuroglía/química , Fragmentos de Péptidos/análisis , ARN Mensajero/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Chronic treatment with converting enzyme inhibitors induces a fall of plasma angiotensinogen concentration, which is thought to result from increased consumption by renin. As this explanation cannot account for all the observations, we reexamined this effect. Rats received captopril (50 mg/kg per day), enalapril (10 mg/kg per day) or Hoe 498 (1 mg/kg per day) for 7 days. Plasma angiotensinogen (by indirect assay) fell to 34, 37 and 43% of its initial values, respectively. Total immunoreactive angiotensinogen (by direct radioimmunoassay, which also measures des-AI-angiotensinogen) was 76, 70 and 95% of the initial values, respectively. This suggests that a major part of the fall of intact angiotensinogen can be attributed to increased cleavage by renin, but an additional factor is likely to be involved. Experiments with isolated hepatocytes indicated that converting enzyme inhibitors in high concentrations may have a direct effect on angiotensinogen secretion. Whether this can account for the fall in total immunoreactive angiotensinogen in vivo remain unclear.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina , Angiotensinógeno/sangre , Angiotensinas/sangre , Angiotensinógeno/metabolismo , Animales , Compuestos Bicíclicos con Puentes/farmacología , Captopril/farmacología , Enalapril/farmacología , Hígado/metabolismo , Masculino , Ramipril , Ratas , Renina/sangre , Equilibrio HidroelectrolíticoAsunto(s)
Gonadotropina Coriónica/farmacología , Lectinas/farmacología , Activación de Linfocitos/efectos de los fármacos , Animales , Depresión Química , Cabras/inmunología , Humanos , Lectinas/antagonistas & inhibidores , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Conejos/inmunología , Timidina/metabolismo , TritioRESUMEN
Rat angiotensinogen (Ao) consists of two molecular weight forms (Ao-1 and Ao-2). The amino acid composition of both forms is very similar. In addition, the immunological properties of both molecules obtained by immunodiffusion and direct radioimmunoassay are very similar as well. Beside the difference in molecular weight there is a heterogeneity in isoelectric points within and between both forms. The difference in the carbohydrate moiety between both forms is the basis of the suggestion that the main difference in weight and charge is due to the presence or absence of one oligosaccharide chain. The twin bands of Ao-2 in IEF which have the same amount of sialic acid, can be separated on ConA Sepharose CL-6B, indicating that different types of oligosaccharide chains are attached to Ao-2. The structural features of angiotensinogen have been studied by circular dichroism. The structure of angiotensinogen consists of 17% alpha-helix, 37% beta-structure and 45% irregular structure. Concentrations of dithiothreitol (DTT) of up to 2o mM cause a decrease in regular structure at neutral pH and an increase in regular structure at pH 9.5. There is no change in the amount of angiotensin I cleaved by the enzyme renin. However the binding of angiotensinogen to a specific rabbit antibody is decreased following treatment with 1o or 2o mM DTT.
Asunto(s)
Angiotensinógeno , Angiotensinas , Aminoácidos/análisis , Animales , Fenómenos Químicos , Química , Cromatografía de Afinidad , Dicroismo Circular , Focalización Isoeléctrica , Masculino , Peso Molecular , Conformación Proteica , Radioinmunoensayo , Ratas , Ratas EndogámicasRESUMEN
The present study investigated the hypothesis that the increase in plasma angiotensinogen after nephrectomy is mediated by endogenous renin and angiotensin (ANG) II. Rats were divided into control, nephrectomy, or nephrectomy plus adrenalectomy groups. In addition, similar cohorts were divided as just mentioned and then given either atenolol (selective beta 1-adrenoceptor inhibitor that prevents renin release) or Dup-753 [ANG II (AT1) receptor antagonist]. The plasma angiotensinogen levels increase approximately fivefold after 24 h in nephrectomized rats. Pretreatment with atenolol blunted this increase. A significant reduction was observed 4 h (P < 0.05) and 8 h (P < 0.005) after surgery. Dup-753 nearly abolished the increase in angiotensinogen plasma levels. After pretreatment with Dup-753, significantly higher angiotensinogen levels (P < 0.005) were found only after 24 h. Nephrectomy plus adrenalectomy also blunted the rise in plasma angiotensinogen. A significant increase in angiotensinogen plasma levels could only be observed after 8 h (P < 0.005) and 12 h (P < 0.005) but not after 24 h. Atenolol further reduced this increase. After atenolol pretreatment, significantly higher angiotensinogen levels could only be observed after 12 h (P < 0.05). Dup-753 completely abolished the increase of plasma angiotensinogen after nephrectomy plus adrenalectomy. In anesthetized control rats at time 0 the plasma ANG I levels were 0.7 nM. Pretreatment with atenolol decreased the ANG I values by 30%, whereas Dup-753 caused a sixfold increase in plasma ANG I levels in the control rats at time 0.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Angiotensina II/fisiología , Angiotensinógeno/biosíntesis , Hígado/metabolismo , Nefrectomía , Adrenalectomía , Angiotensinógeno/sangre , Animales , Atenolol/farmacología , Compuestos de Bifenilo/farmacología , Imidazoles/farmacología , Riñón/metabolismo , Losartán , Masculino , Nefrectomía/métodos , Concentración Osmolar , Periodo Posoperatorio , Ratas , Ratas Wistar , Renina/sangre , Renina/metabolismo , Tetrazoles/farmacología , Factores de TiempoRESUMEN
Because all antibodies to bradykinin described to date more or less cross-react with larger and smaller bradykinin derivatives, the aim of the present study was the induction of a specific antibody against bradykinin. A bradykinin derivative, containing a Cys residue at position 6 instead of Ser, was linked to BSA by using a new heterobifunctional cross-linking reagent, the N-maleimido-6-aminocaproyl ester of 1-hydroxy-2-nitro-4-benzenesulfonic acid (mal-sac-HNSA). The Cys6-bradykinin derivative conjugated via the SH group to BSA was used to elicit bradykinin-specific antibodies in rabbits. After the fifth booster injection, the cross-reactivity of this antiserum with kallidin is 4 X 10(-3) when compared with bradykinin. The cross-reactivity of the de-Arg1-bradykinin derivative was 2 X 10(-4), indicating that the antibody requires the free N-terminus. The cross-reactivity of bradykinin and de-Arg9-bradykinin was nearly identical. However, de-Arg9-de-Phe8-bradykinin has a binding of 6 X 10(-3). From these data, it can be concluded that due to the ring-like structure of bradykinin, the coupling of bradykinin in the center of the molecule will elicit an antiserum that needs both the free N-terminal as well as the C-terminal phenylalanine and arginine residues for antibody recognition.